Preterm birth and mechanical ventilation (MV) trigger a pulmonary response. The response results in neonatal chronic lung disease (CLD), characterized by alveolar simplification. However, the mechanisms through which prematurity and MV combine to dysregulate alveolar formation are not well defined. A potential mechanism is epigenetics, which uses histone covalent modifications and DNA CpG methylation to regulate gene transcription. Premature lambs develop neonatal CLD when managed by MV but not high-frequency nasal ventilation (HFNV). Our other novel discoveries are that MV causes pulmonary 1) genome-wide hypoacetylation and hypermethylation of histone 3 (H3); 2) increased expression of insulin-like growth factor-1 (IGF-1); and 3) disruption of the histone code along the IGF-1 gene locus. Our focus on the regulation of IGF-1 expression is relevant because preterm humans with neonatal CLD have increased pulmonary IGF-1 expression. Another novel discovery is that these epigenetic changes do not happen with HFNV. Exciting new data suggest that two interventions (histone deacetylase [HDAC] inhibitors; vitamin A+retinoic acid, VARA) during MV improve alveolar formation and change epigenetic characteristics. These new data suggest that epigenetics participate in neonatal CLD. Our central hypothesis is that preterm birth and MV dysregulate histone covalent modifications and DNA CpG methylation in the lung compared to HFNV in a well-established animal model of neonatal CLD. Because the focus of our proposal is epigenetic mechanisms, we will also use HDAC inhibitors or VARA) during MV to inhibit the dysregulation caused by MV alone on pulmonary epigenetics, histology, respiratory gas exchange, and pulmonary mechanics. Specific Aim 1 will determine whether MV causes genome-wide dysregulation of H3 covalent modifications and DNA methylation in the lung. Specific Aim 2 will determine whether MV dysregulates IGF-1 histone covalent modifications and DNA methylation at functionally important regions that determine gene recognition sites and gene transcription initiation, exon selection, elongation, and termination. Specific Aim 3 will determine whether increased pulmonary IGF-1 expression during MV contributes to alveolar simplification and poor lung function. Our proposal is innovative because it tests the hypothesis that preterm birth and MV change epigenetic characteristics and alveolar formation in parallel. Our study design drills-down from pulmonary genome-wide histone modifications and DNA CpG methylation to the histone code along the IGF-1 gene locus, and relates the results to alveolar formation, respiratory gas exchange, and lung mechanics. Our track record provides confidence that we can accomplish these complex and long studies, in which we will use our unique large-animal, physiological model of neonatal CLD. We will use IGF-1 as a paradigm because of our unique expertise on IGF-1 epigenetics. The significance of our proposal is that it has the potential to shift the paradigm about both the molecular and physiologic pathogenesis of neonatal CLD.